Synaptogenesis is a developmental process fundamental to the establishment of neuronal networks. However, there is relatively little knowledge of the electrophysiologically detectable events occurring during synaptogenesis, and little knowledge concerning the basic mechanisms that regulate whether a synapse will form between two neurons. In fact much of our current thinking has arisen from the neuromuscular junction. The aims of the proposed research are to study synaptogenesis between neuron pairs and to investigate a specific synaptogenic regulatory mechanism. The hypothesis that "the formation of a chemical synapse requires the cessation of neurite extension in the presynaptic neuron" will be tested here. Using identified neurons of the pulmonate mollusc Helisoma it will be possible to gain a level of precision essential to such a study. Identified neurons can readily be plated into culture as specific neuron pairs where they will form previously characterized chemical synaptic connections. Furthermore, in cell culture it is possible to make direct experimental observations and manipulations. Thus, in testing this hypothesis, the timing of the cessation of neurite extension will be correlated with the timing of the formation of chemically synapses. Additionally, the cessation of neurite extension will be experimentally manipulated. The neurotransmitters serotonin and dopamine cause a premature cessation of neurite extension. Consequently these agents will be added to culture media to determine if synaptic connections form parrallel with our experimentally evoked inhibition of neurite extension. Such experiments take strides towards elucidating regulatory mechanisms controlling the formation of synaptic connections. To more directly investigate potential mechanisms that can regulate the formation of the synapse a higher level of resolution will also be utilized. In these studies single synaptic terminals will be voltage clamped at various times in the development of synaptic connections to determine whether the appearence of presynaptic ionic conductances may regulate the development of functional synaptic transmission. Thus, this proposal seeks to elucidate regulatory mechanisms that control the formation of chemical synapses, and to investigate such mechanisms at a new level of resolution by direct studies at single synaptic terminals.